Identification of the HetR Recognition Sequence Upstream of hetZ in Anabaena sp. Strain PCC 7120

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Abstract
HetR is the master regulator of heterocyst differentiation in Anabaena sp. strain PCC 7120 and has been found to specifically bind to an inverted-repeat-containing region upstream of hetP, a heterocyst differentiation gene. However, no such inverted-repeat sequence can be found in promoters of other genes in the genome. hetZ is a gene involved in early heterocyst differentiation. As shown with the gfp reporter gene, transcription from PhetZ was correlated to the expression level of hetR and inhibition by RGSGR, the pentapeptide derived from the C terminus of PatS. As detected by electrophoretic mobility shift assay, a recombinant HetR showed specific binding to the region upstream of hetZ, and the binding was inhibited by RGSGR. Tests of a series of the upstream fragments delimited the HetR-binding site to a 40-bp region that shows similarity to that upstream of hetP. The introduction of substitutions of bases conserved in the two HetR-binding sites showed that at least 12 bases are required for recognition by HetR. Deletion of a 51-bp region containing the HetR-binding site completely eliminated the transcription activity of PhetZ. Based on the HetR recognition sequence of hetZ, those upstream of hetR and patA are proposed.